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The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.
Cyanobacteria are prokaryotic organisms characterised by their complex structures and a wide range of pigments. With their ability to fix CO2, cyanobacteria are interesting for white biotechnology as cell factories to produce various high-value metabolites such as polyhydroxyalkanoates, pigments, or proteins. White biotechnology is the industrial production and processing of chemicals, materials, and energy using microorganisms. It is known that exposing cyanobacteria to low levels of stressors can induce the production of secondary metabolites. Understanding of this phenomenon, known as hormesis, can involve the strategic application of controlled stressors to enhance the production of specific metabolites. Consequently, precise measurement of cyanobacterial viability becomes crucial for process control. However, there is no established reliable and quick viability assay protocol for cyanobacteria since the task is challenging due to strong interferences of autofluorescence signals of intercellular pigments and fluorescent viability probes when flow cytometry is used. We performed the screening of selected fluorescent viability probes used frequently in bacteria viability assays. The results of our investigation demonstrated the efficacy and reliability of three widely utilised types of viability probes for the assessment of the viability of Synechocystis strains. The developed technique can be possibly utilised for the evaluation of the importance of polyhydroxyalkanoates for cyanobacterial cultures with respect to selected stressor-repeated freezing and thawing. The results indicated that the presence of polyhydroxyalkanoate granules in cyanobacterial cells could hypothetically contribute to the survival of repeated freezing and thawing.
- MeSH
- fluorescence MeSH
- fluorescenční barviva * metabolismus chemie MeSH
- fyziologický stres * MeSH
- mikrobiální viabilita * MeSH
- polyhydroxyalkanoáty metabolismus MeSH
- průtoková cytometrie * MeSH
- sinice metabolismus fyziologie MeSH
- Synechocystis * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface in vitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.
Halophilic bacteria are extremophiles that thrive in saline environment. Their ability to withstand such harsh conditions makes them an ideal choice for industrial applications such as lignocellulosic biomass degradation. In this study, a halophilic bacterium with the ability to produce extracellular cellulases and hemicellulases, designated as Nesterenkonia sp. CL21, was isolated from mangrove sediment in Tanjung Piai National Park, Malaysia. Thus far, studies on lignocellulolytic enzymes concerning bacterial species under this genus are limited. To gain a comprehensive understanding of its lignocellulose-degrading potential, the whole genome was sequenced using the Illumina NovaSeq 6000 platform. The genome of strain CL21 was assembled into 25 contigs with 3,744,449 bp and a 69.74% GC content and was predicted to contain 3,348 coding genes. Based on taxonomy analysis, strain CL21 shares 73.8 to 82.0% average nucleotide identity with its neighbouring species, below the 95% threshold, indicating its possible status as a distinct species in Nesterenkonia genus. Through in-depth genomic mining, a total of 81 carbohydrate-active enzymes were encoded. Among these, 24 encoded genes were identified to encompass diverse cellulases (GH3), xylanases (GH10, GH11, GH43, GH51, GH127 and CE4), mannanases (GH38 and GH106) and pectinases (PL1, PL9, and PL11). The production of lignocellulolytic enzymes was tested in the presence of several substrates. This study revealed that strain CL21 can produce a diverse array of enzymes which are active at different time points. By combining experimental data with genomic information, the ability of strain CL21 to produce lignocellulolytic enzymes has been elucidated, with potential applications in biorefinery industry.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- celulasy genetika metabolismus MeSH
- fylogeneze * MeSH
- genom bakteriální * MeSH
- genomika * MeSH
- geologické sedimenty mikrobiologie MeSH
- glykosidhydrolasy * genetika metabolismus MeSH
- lignin * metabolismus MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenování celého genomu MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
... TYPU 44 -- 4.1 Utilizace energetických substrátů při sportu 44 -- 4.2 Hormonální regulace metabolismu ...
Jessenius
Třetí aktualizované a doplněné vydání 227 stran : barevné ilustrace ; 20 cm
Příručka, která se zaměřuje na sportovní aktivitu u pacientů s diabetem mellitus. Obsahuje i kazuistiky. Určeno odborné veřejnosti.; Diabetes u dětí a mladistvých byl tradičně považován za překážku větší sportovní aktivity. Dramatické zlepšení kvality života diabetiků 1. typu – v důsledku nových technických i farmakologických možností inzulinové terapie – umožňuje mnohým z nich život téměř srovnatelný s jejich zdravými vrstevníky. Přirozeným zájmem se tak mezi diabetiky 1. typu stává sport, včetně jeho závodního provozování. Diabetolog dnes musí být schopen pečovat o aktivně sportujícího diabetika. Riziko hypoglykemie i další nebezpečí spojená se sportem nelze podceňovat, na druhou stranu se lékař dostává do nepříjemného světla, když mladému pacientovi sport zakáže, když on sám zná další diabetiky, kteří se sportu věnovat mohou, a to někdy i vrcholovému. Třetí vydání úspěšné publikace obsahuje i nejmodernější metody léčby a kontroly glykemie, tedy aplikaci inzulinových pump a okamžitého měření glukózy (CGM). Knížka kolektivu autorů vedených jedním z nejuznávanějších českých odborníků v oblasti diabetologie a výživy, prof. MUDr. Zdeňkem Rušavým, Ph.D., je koncipována jako praktický návod pro diabetologa, resp. ošetřujícího lékaře, který se stará o sportující diabetiky 1. typu.
- MeSH
- diabetes mellitus MeSH
- selfmonitoring glykemie MeSH
- sportovci MeSH
- sporty MeSH
- tělesná námaha MeSH
- tělesná výkonnost MeSH
- Publikační typ
- kazuistiky MeSH
- příručky MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- diabetologie
- tělovýchovné lékařství
- NLK Publikační typ
- kolektivní monografie
Stereoselective synthesis of spirocyclic compounds containing heterocyclic motifs represents a formidable challenge in enantioselective synthesis. Here, we present a cascade reaction between α,β-unsaturated aldehydes and isoxazolones under synergistic catalysis of a chiral secondary amine and a palladium(0) catalyst. This strategy allows access to chiral spiroisoxazolone derivatives with a large substrate scope tolerance and high levels of diastereoselectivity (dr up to 20:1) and enantioselectivity (up to 99% ee). Furthermore, the utility of this methodology is showcased by the transformation of chiral spiroisoxazolones into structurally attractive and enantiomerically enriched cyclopentene carboxylic acids with two stereogenic centers.
- Publikační typ
- časopisecké články MeSH
We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing cationic substituents (protonated amino, methylamino, dimethylamino and trimethylammonium groups) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through hex-1-ynyl or propargyl linker. These cationic dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase. In primer extension (PEX), we successfully obtained DNA containing one, two, three, or (all) four modified nucleotides, each bearing a different cationic modification. The cationic dNTPs were somewhat worse substrates compared to previously studied dNTPs bearing hydrophobic or anionic modifications, but the polymerase was still able to synthesize sequences up to 73 modified nucleotides. We also successfully combined one cationic modification with one anionic and two hydrophobic modifications in PEX. In polymerase chain reaction (PCR), we observed exponential amplification only in the case of one cationic modification, while the combination of more cationic nucleotides gave either very low amplification or no PCR product. The hypermodified oligonucleotides prepared by PEX were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies of hybridization, denaturation, and circular dichroism spectroscopy showed that the presence of cationic modifications increases the stability of duplexes.
For the treatment of bilateral limbal stem cell deficiency (LSCD), cell therapy with transplantation of cultivated oral mucosa epithelial cells (COMET) is a promising alternative. Although not yet established, current protocols on the cultivation of oral mucosal epithelial cell (OMECs) sheets are based mainly on substrates and xenobiotic additives that may lead to variable outcomes and undesirable immune responses by the patient. The aim of this study was to characterize OMECs cultivated in xenobiotic-free media (XF) seeded on fibrin gel, in comparison to conventional complex (COM) medium. Oral mucosal biopsies were retrieved from 31 donors. After cultivation in COM or XF medium, OMECs were compared based on growth kinetics, morphology, cell size and viability. Using immunofluorescence and gene expression analyses, the degree of stemness, proliferation and differentiation was evaluated in OMEC cultures. Our findings showed that although OMECs showed a similar morphology and viability, and comparable growth kinetics, immunofluorescence revealed the preservation of stemness (p63 + p40 positivity in cells ≤11 μm) and proliferation in both COM and XF. Gene expression analyses showed that keratin (K)13 and K15 expression levels were significantly higher in XF (adj. p < 0.001), but otherwise COM and XF-treated OMECs had comparable transcriptional profiles in a panel of stemness, proliferation and differentiation genes. These results demonstrate the feasibility of culturing OMECs on fibrin gel without xenogeneic additives, while maintaining their undifferentiated state and preserving stemness. In conclusion, both in terms of results and methodology, the procedures presented here are suitable for implementation in clinical practice.
- MeSH
- buněčná diferenciace MeSH
- buněčné kultury * MeSH
- deficit limbálních kmenových buněk MeSH
- dospělí MeSH
- epitelové buňky * metabolismus účinky léků MeSH
- fibrin * MeSH
- gely MeSH
- kmenové buňky * metabolismus cytologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- limbus corneae * cytologie patologie metabolismus MeSH
- nemoci rohovky patologie farmakoterapie metabolismus MeSH
- proliferace buněk účinky léků MeSH
- rohovkový epitel metabolismus cytologie účinky léků patologie MeSH
- senioři MeSH
- transplantace kmenových buněk metody MeSH
- ústní sliznice * cytologie MeSH
- viabilita buněk MeSH
- xenobiotika farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Measuring the transduction of electrical signals within neurons is a key capability in neuroscience. Fluorescent voltage sensitive dyes (VSDs) were early tools that complemented classical electrophysiology by enabling the optical recording of membrane potential changes from many cells simultaneously. Recent advances in the VSD field have led to bright and highly sensitive sensors that can be targeted to the desired cell populations in live brain tissue. Despite this progress, recently, protein-based genetically encoded voltage indicators (GEVIs) have become the go-to tools for targeted voltage imaging in complex environments. In this Perspective, we summarize progress in developing targetable VSDs, discuss areas where these synthetic sensors are or could become relevant, and outline hurdles that need to be overcome to promote the routine use of targetable VSDs in neuroscience research.
- MeSH
- akční potenciály fyziologie účinky léků MeSH
- fluorescenční barviva MeSH
- lidé MeSH
- membránové potenciály fyziologie MeSH
- neurony * fyziologie MeSH
- zobrazování pomocí barviva citlivého na potenciál metody trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
Microbial diversity plays a crucial role in litter decomposition. However, the relationships between microbial diversity and substrate successional stage are the drivers of this decomposition. In this study, we experimentally manipulated microbial diversity and succession in post-mining soil. We used leaf litter samples from two forests of a post-mining site near Sokolov, Czech Republic: one alder plantation and one mixed forest with birch aspen and willow. Litter from each site was decomposed in the field for 3 and 12 months. The litter was X-ray sterilized and part of the litter was kept unsterilized to produce inoculum. Leaf litter samples of two different ages (3 and 12 months) from each site were each inoculated with litter of two different ages (3 and 12 months), using less and more diluted inoculum, producing two levels of microbial diversity. In each of these eight treatments, the bacterial community was then characterized by amplicon sequencing of the 16S rRNA gene and microbial respiration was used to assess the rate of decomposition. A significantly higher respiration (p < 0.05) was found for the litter inoculated with the higher level of microbial diversity. Higher respiration was also found for the younger litter compared to the older litter and both litter origins. This shows a reduction in microbial respiration with substrate age and inoculation diversity, suggesting that microbial diversity supports the decomposition of soil organic matter.
- Publikační typ
- časopisecké články MeSH
